Studies on cell cycle traverse, DNA synthesis, and cell proliferation have been significantly advanced through the use of labeled DNA precursors. More recently, nonradioactive molecules, such as the base analoque 5 BrdU, have been substituted for radio-labeled DNA precursors. BrdU is substituted stoichiometrically for thymidine in DNA during the S-phase (synthesis) of cell growth where the cellular DNA content is doubling between the G.sub.1 and G.sub.2 cell phases. A measure of the incorporated BrdU is related to the new DNA content, i.e., the status or location of the cell in the S-phase.
Thus, the detection of BrdU-substituted DNA is functionally related to synthesis of DNA during the S-phase of the cell cycle. One detection technique employs an immunofluorescent assay of BrdU. This technique requires the partial denaturation of cellular DNA by heat or acid treatment to expose the incorporated BrdU to the antibody. In a variation of the procedure, a fluorescent counterstain, propidium iodide (PI), is included to measure the total DNA content. Using two-color, flow cytometric analysis, cells containing incorporated BrdU are readily detected and their cell cycle position is easily assessed.
Another method for detecting BrdU-substituted DNA uses the A-T base binding fluorochrome, Hoechst 33258, that is quenched when bound to A-BrdU regions in double-stranded DNA. This effect is described in Samuel A. Latt, "Microfluorometric Detection of Deoxyribonucleic Acid Replication in Human Metaphase Chromosomes," Proc. Nat. Acad. Sci. USA 70 No. 12, p. 3395-3399 (December 1973). The BrdU/Hoechst fluorescence quenching technique, however, is not as sensitive as the immunofluorescent assay. There is difficulty in assessing quantitatively the reduction of Hoechst fluorescence after short exposure periods to BrdU, e.g., 30 minutes. BrdU exposure periods of six hours or longer were required for cells to incorporate sufficient amounts of BrdU for assessing Hoechst quenching.
The present invention overcomes the problems of resolution and sensitivity with conventional Hoechst quenching processes and an improved process is provided for detecting BrdU/Hoechst fluorescence quenching.
Accordingly, one object of the present invention is to provide a sensitive technique for enhancing the detection of Hoechst quenching in labeled DNA.
Another object of the present invention is to enable the quantitation of BrdU during cell S-phase growth.
One other object of the present invention is to provide an assay process for BrdU which minimizes cell loss and damage to cellular markers in DNA, RNA, and/or chromatin.